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Indole-Containing Phytoalexin-Based Bioisosteres As Antifungals: in Vitro and in Silico Evaluation Against Fusarium Oxysporum

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Indole-Containing Phytoalexin-Based Bioisosteres As Antifungals: in Vitro and in Silico Evaluation Against Fusarium Oxysporum molecules Article Indole-Containing Phytoalexin-Based Bioisosteres as Antifungals: In Vitro and In Silico Evaluation against Fusarium oxysporum Andrea Angarita-Rodríguez, Diego Quiroga and Ericsson Coy-Barrera * Bioorganic Chemistry Laboratory, Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, Campus Nueva Granada, Cajicá 250247, Colombia; [email protected] (A.A.-R.); [email protected] (D.Q.) * Correspondence: [email protected]; Tel.: +57-1-6500000 (ext. 3270) Academic Editors: Maria Daglia, Simone Carradori and Annabella Vitalone Received: 18 November 2019; Accepted: 20 December 2019; Published: 21 December 2019 Abstract: There is a continuous search for more reliable and effective alternatives to control phytopathogens through different strategies. In this context, indole-containing phytoalexins are stimuli-induced compounds implicated in plant defense against plant pathogens. However, phytoalexins’ efficacy have been limited by fungal detoxifying mechanisms, thus, the research on bioisosteres-based analogs can be a friendly alternative regarding the control of Fusarium phytopathogens, but there are currently few studies on it. Thus, as part of our research on antifungal agents, a set of 21 synthetic indole-containing phytoalexin analogs were evaluated as inhibitors against the phyopathogen Fusarium oxysporum. Results indicated that analogs of the N,N-dialkylthiourea, N,S-dialkyldithiocarbamate and substituted-1,3-thiazolidin-5-one groups exhibited the best docking scores and interaction profiles within the active site of Fusarium spp. enzymes. Vina scores exhibited correlation with experimental mycelial growth inhibition using supervised statistics, and this antifungal dataset correlated with molecular interaction fields after CoMFA. Compound 24 (tert-butyl (((3-oxo-1,3-diphenylpropyl)thio)carbonothioyl)-l-tryptophanate), a very active analog against F. oxysporum, exhibited the best interaction with lanosterol 14α-demethylase according to molecular docking, molecular dynamics and molecular mechanic/poisson-boltzmann surface area (MM/PBSA) binding energy performance. After data analyses, information on mycelial growth inhibitors, structural requirements and putative enzyme targets may be used in further antifungal development based on phytoalexin analogs for controlling phytopathogens. Keywords: phytopathogens; Fusarium spp.; antifungals; indole-containing analogs 1. Introduction Fungi from the genus Fusarium are very important because they have wide cosmopolitan-type geographic distribution and high relevance for agriculture and economy [1]. Their diversity is quite common in soils and many of them have the capacity to cause infectious diseases in different types of crop plants. Additionally, some species can cause opportunistic infections in animals due to the production of toxins that can affect them [1]. Genus Fusarium gathered phytopathogenic-behaved species causing necrosis-associated diseases in several plants around the world. This fungus can survive in the soil in mycelial or conidial forms during host absence. When a host is present, the mycelium penetrates its roots, enters the vascular system (xylem) to move and multiply itself, causing wilt symptoms. Soil-borne fungi Fusarium spp. can infects the host by using different sets of genes for early host-plant signaling, as well as the binding and enzymatic decomposition of antifungal compounds produced by plant for defense, in order to inactivate and kill host cells by Molecules 2020, 25, 45; doi:10.3390/molecules25010045 www.mdpi.com/journal/molecules Molecules 2020, 25, 45 2 of 19 fungal toxins [2]. In this context, some control strategies based on chemical, physical and cultural methods have arisen to mitigate the negative effects of this phytopathogen to host plants. Chemical treatments include formaldehyde applications to avoid disseminations as well as dazomet, sodium methan, methyl isothiocyanate and systemic fungicides as benomyl, thiabendazon, carbendazim and methylthiophanate [3]. Fusarium-caused diseases treatment by systemic fungicides, although it has good effectivity, is also problematic, since these kind of treatments can become mutagenics for several plants and these agents can also generate high degrees of resistance [3,4]. Plants involve a systemic defense mechanism involving some metabolites such as phytoalexins. These metabolites are synthesized in adjacent areas of healthy cells to those damaged cells and they are accumulated both in necrotic and susceptible resistant tissues [5]. In other words, they are strictly produced in a site around the place where infection needs to be controlled. Thus, the resistance occurs when one or more phytoalexins reach a high enough concentration to inhibit the pathogen development [5]. However, some phytopathogens are recently described to exhibit strategies to invade the plant tissues and obtain necessary nutrients for its growing and reproduction [1,2,4]. Despite that plants are capable to synthesize specific antifungal metabolites, the fungi are capable to produce particular enzymes in order to detoxify quickly and inhibit the phytoalexin activity. This detoxifying capacity is described by Pedras et al. [6] as an “arms race” that advantaged the pathogen, causing adverse effects in the crop. An example of the previous fact is the case of brassinin (an important phytoalexin for crucifers due its dual potential as an antimicrobial defense and a biosynthetic precursor of phytoalexin variety), since if this metabolite is inhibited by a fungal enzyme, the plant become possibly more susceptible to be attacked by the phytopathogen [6]. Thus, the cruciferous invader L. maculans (a devastating pathogen) can generate itself a detoxification defense against phytoalexins. The mechanism is based on brassinin oxidase production, which catalyzes the brassinin oxidation through the dithiocarbamate transformation into an aldehyde. This mechanism depends on direct brassinin recognition by this enzyme. Therefore, exploration of brassinin analogs acting as bioisosteres can be adopted as an important approach for the development of novel antifungals [7]. In this sense, bioisosterism is generally a chemical strategy for the rational design of bioactives from structural modifications of leads, whose action mechanism and chemical structure is therefore known. From the above fact, it is possible to develop novel compounds with chemical and/or physical similarities but producing identical or even better biological properties [7]. Therefore, as part of our research on antifungals discovery and development, the present work was initially focused on the in silico exploration, by molecular docking, on the interaction of a set of indole-containing phytoalexin analogs within the active site of 25 enzymes reported to Fusarium spp. Subsequently, the integration of the experimental antifungal activity (i.e., Fusarium oxysporum mycelial growth inhibition) and docking scores datasets (by supervised multivariate statistics) as well as the three-dimensional (3D) molecular descriptors and antifungal activity datasets (on the basis of 3D quantitative structure-activity relationship (3D-QSAR) by comparative molecular field analysis (CoMFA)) led to recognize some important hits, enzyme targets and structural requirements to be stated for the further development of antifungals for controlling phytopathogens using phytoalexin-like analogs. Finally, some insights into the binding mode of a 3-oxo-1,3-diphenylpropyl N-alkyldithiocarbamate-type analog and the homology model of the transmembrane F. cerealis lanosterol 14-demethylase were also determined in order to rationalize the antifungal activity of this type of synthetic phytoalexin analogs. 2. Results and Discussion 2.1. Test Indole-Containing Phytoalexin Analogs and Docking Protocol Validation A set of 25 synthetic indole-containing compounds (1–25) (Figure1) were obtained by our previously reported reaction route from l-tryptophan [8] (Figure S1). Their IUPAC names are displayed in the complementary material (Table S1). Such compounds were then chosen as test compounds owing to their antifungal activity by inhibiting mycelial growth of F. oxysporum [8,9]. Thus, in order to explore Molecules 2020, 25, 45 3 of 19 Molecules 2019, 24, x FOR PEER REVIEW 3 of 19 andThus, outline in order a further to explore information and outline related a to further the potential information and putative related targets to the of potential these compounds and putative at in silicotargets level, of these docking compounds simulations at in were silico therefore level, do performedcking simulations with 25 fungalwere therefore enzymes performed (E1–E25) (Table with A1 25 (Appendixfungal enzymesA)), which (E1–E25) were (Table selected A1), due which to their were significant selected associations due to their on significant pathogenic associations and metabolic on performancepathogenic and of Fusarium metabolicspp. performance of Fusarium spp. FigureFigure 1. 1. StructuralStructural classification classification of the of test the indo testle-containing indole-containing phytoalexin phytoalexin analogs: analogs:2-alkyl 2-alkylaminoesters aminoesters (1–4), N (,1N–4-dialkylthioureas), N,N-dialkylthioureas (5–8), 2-cyanoethyl (5–8), 2-cyanoethyl N-alkyldithiocarbamatesN-alkyldithiocarbamates (9–12), (3-methoxy-3-oxopropyl9–12), 3-methoxy-3-oxopropyl N-alkyldithiocarbamateN-alkyldithiocarbamate (13 (–1316–),16 ),2-methyl-4-oxopentan-2-yl NN-alkyldithiocarbamate-alkyldithiocarbamate
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